Her2 binders

ABSTRACT

Imaging agents comprising an isolated polypeptide conjugated with a radionucleide and a chelator; wherein the isolated polypeptide binds specifically to HER2, or a variant thereof; and methods for preparing and using these imaging agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/083,424, entitled “HER2 BINDERS,” filed on Mar. 29, 2016, whichitself is a divisional of U.S. patent application Ser. No. 12/975,425,entitled “HER2 BINDERS,” filed on Dec. 22, 2010, now abandoned, whichitself is a continuation in part of U.S. patent application Ser. No.12/735,068, entitled “Polypeptides having affinity for HER2,” now U.S.Pat. No. 8,501,909, filed on Oct. 6, 2010, which is the national stageof international application PCT/EP2008/068167, filed on Dec. 22, 2008,the disclosure of all of which are incorporated herein by reference intheir entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 13, 2010, isnamed 2355971.txt and is 4,957 bytes in size.

FIELD

The invention relates generally to imaging agents that bind to humanepidermal growth factor receptor type 2 (HER2) and methods for makingand using such agents.

BACKGROUND

Human epidermal growth factor receptor type 2 (HER2) is a transmembraneprotein and a member of erbB family of receptor tyrosine kinaseproteins. HER2 is a well-established tumor biomarker that isover-expressed in a wide variety of cancers, including breast, ovarian,lung, gastric, and oral cancers. Therefore, HER2 has great value as amolecular target and as a diagnostic or prognostic indicator of patientsurvival, or a predictive marker of the response to antineoplasticsurgery.

Over the last decade, noninvasive molecular imaging of HER2 expressionusing various imaging modalities has been extensively studied. Thesemodalities include radionuclide imaging with Positron EmissionTomography (PET) and Single Photon Emission Tomography (SPECT). PET andSPECT imaging of HER2 (HER2-PET and HER2-SPECT, respectively) providehigh sensitivity, high spatial resolution. PET imaging of HER2 alsoprovides strong quantification ability. HER2-PET and HER2-SPECT areparticularly useful in real-time assays of overall tumor HER2 expressionin patients, identification of HER2 expression in tumors over time,selection of patients for HER-targeted treatment (e.g.,trastuzumab-based therapy), prediction of response to therapy,evaluation of drug efficacy, and many other applications. However, noPET or SPECT-labeled HER2 ligands have been developed that have achemistry and exhibit in vivo behaviors which would be suitable forclinical applications.

Naturally occurring Staphylococcal protein A comprises domains that forma three-helix structure (a scaffold) that binds to the fragment,crystallizable region (Fc) of immunoglobulin isotype G (IgG). Certainpolypeptides, derived from the Z-domain of protein A, contain a scaffoldcomposed of three α-helices connected by loops. Certain amino acidresidues situated on two of these helices constitute the binding sitefor the Fc region of IgG. Alternative binder molecules have beenprepared by substituting surface-exposed amino acid residues (13residues) situated on helices 1 and 2, to alter the binding ability ofthese molecules. One such example is HER2 binding molecules or HER2binders. These HER2 binders have been labeled with PET or SPECT-activeradionuclides. Such PET and SPECT-labeled binders provide the ability tomeasure in vivo HER2 expression patterns in patients and would thereforeaid clinicians and researchers in diagnosing, prognosing, and treatingHER2-associated disease conditions.

HER2 binding affibody molecules, radiolabeled with the PET-activeradionucleide, ¹⁸F, have been evaluated as imaging agents for malignanttumors that over express HER2. HER2 binding Affibody molecules,conjugated with ^(99m)Tc via the chelators such as maGGG(mercaptoacetyltriglycyl), CGG (cysteine-diglycyl), CGGG (SEQ ID NO: 6)(cysteine-triglycyl) or AA3, have also been used for diagnostic imaging.The binding of these molecules to target HER2 expressing tumors has beendemonstrated in mice.

In most of the cases, the signal-generating ¹⁸F group is introduced tothe Affibody through a thiol-reactive maleimide group. The thiolreactive maleimide group is prepared using a multi-step synthesis after¹⁸F incorporation. However, this chemistry only provides a lowradiochemical yield. Similarly, the conjugation of ^(99m)Tc with theAffibody is a multistep, low yield, process. In addition, Tc reductionand the complex formation with chelates, require high pH (e.g., pH=11)conditions and long reaction times.

Though the in vivo performance of ¹⁸F labeled Affibody molecules wasmoderately good, there is significant room for improvement. For example,in some studies, the tumor uptake was found to be only 6.36±1.26% ID/g 2hours post-injection of the imaging agent. On the other hand, ^(99m)Tclabeled Affibody molecules have predominant hepatobiliary clearance,which causes a high radioactivity accumulation in the intestine, whichrestricts its use for detecting HER2 tumors and metastates in theabdominal area.

Therefore, there is a need for chemistries and methods for synthesizingradiolabeled polypeptides in which the radioactive moiety, such as ¹⁸Fcan be introduced at the final stage, which in turn will provide highradiochemical yields. In addition, there is a need for a new HER2 basedimaging agent for PET or SPECT imaging with improved propertiesparticularly related to renal clearance and toxicity effects.

BRIEF DESCRIPTION

The compositions of the invention are a new class of imaging agents thatare capable of binding specifically to HER2 or variants thereof.

In one or more embodiments, the imaging agent composition comprises anisolated polypeptide comprising SEQ. ID No. 1, SEQ. ID. No 2 or aconservative variant thereof, conjugated with a ^(99m)Tc via adiaminedioxime chelator. The diaminedioxime chelator may comprise Pn216,cPn216, Pn44, or derivatives thereof. The isolated polypeptide bindsspecifically to HER2 or variants thereof.

In one or more embodiments, the imaging agent composition comprises anisolated polypeptide comprising SEQ. ID No. 1, SEQ. ID. No 2 or aconservative variant thereof, conjugated with ⁶⁷Ga or ⁶⁸Ga via a NOTAchelator. The isolated polypeptide binds specifically to HER2 orvariants thereof.

In one or more embodiments, the imaging agent composition comprises anisolated polypeptide comprising SEQ. ID No. 1, SEQ. ID. No 2 or aconservative variant thereof, conjugated with ¹⁸F via a linker. Thelinker comprises a group derived from an aminoxy group, an azido group,or an alkyne group. The isolated polypeptide binds specifically to HER2or variants thereof.

An example of the methods of the invention, for preparing an imagingagent composition, comprises (i) providing an isolated polypeptidecomprising SEQ. ID No. 1, SEQ. ID No. 2 or a conservative variantthereof; and (ii) reacting a diaminedioxime chelator with thepolypeptide to form a chelator conjugated polypeptide. In anotherexample, the method comprises (i) providing an isolated polypeptidecomprising SEQ. ID No. 1, SEQ. ID No. 2 or a conservative variantthereof; (ii) reacting the polypeptide with a linker; and (iii) reactingthe linker with an ¹⁸F moiety to form a ¹⁸F conjugated polypeptide. Thelinker may comprise an aminoxy group, an azido group, or an alkynegroup.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying figures wherein:

FIGS. 1A and 1B are graphs of the surface plasmon resonance (SPR) of thebinding affinity of two anti-HER2 polypeptides, Z477 (SEQ. ID No. 3) and(Z477)₂ (SEQ. ID No. 5), respectively, at eight differentconcentrations, to human HER2.

FIG. 2A and FIG. 2B are graphs of the qualitative flow cytometry of C6(rat glioma, control) and human anti-HER2 antibody to SKOV3 (humanovarian carcinoma) respectively. FIG. 2C shows a bar chart for Her2receptors per cell for SKOV3 and C6 cell lines.

FIG. 3 is a bar graph of ELISA assays for Her2 with respect to a panelof tumor types SKOV3 2-1, SKOV3 3-1, SKOV3 3-4, with respect to SKOV3cells, and blank.

FIG. 4 is a reverse phase HPLC gamma chromatogram of ^(99m)Tc labeledZ00477 (SEQ. ID No. 3).

FIG. 5A is a size exclusion HPLC gamma chromatogram of aggregated^(99m)Tc(CO)₃(His6)Z00477 (SEQ. ID. No. 4) (‘His6’ disclosed as SEQ IDNO: 7) at pH 9. FIG. 5B a size exclusion HPLC gamma chromatogram of nonaggregated ^(99m)Tc(CO)₃(His6)Z00477 (‘His6’ disclosed as SEQ ID NO: 7)labeled affibody standard.

FIG. 6 is a graph of biodistribution profile of Z00477 (SEQ. ID No. 3)in blood, tumor, liver, kidney and spleen samples from SKOV3 tumorbearing mice, including the tumor:blood ratio over time.

FIG. 7 is a diagram of the chemical structure for a Mal-cPN216 linker.

FIG. 8A is a graph of the electrospray ionization time of flight massspectrum (ESI-TOF-MS) and FIG. 8B is a graph of mass deconvolutionresult for the purified Z00477 (SEQ. ID No. 3)-cPN216.

FIG. 9 is a reverse phase HPLC gamma trace chromatogram forZ02891-cPN216 (SEQ. ID No. 2) labeled with ^(99m)Tc.

FIG. 10 is a graph of the biodistribution profile of Z02891 (SEQ. ID No.2) labeled with ^(99m)Tc via cPN216 (% ID, % injected dose)) in blood,liver, kidneys, spleen, and tail samples from SKOV3 tumor bearing mice.

FIG. 11 is a graph of the biodistribution profile of Z02891 (SEQ. ID No.2) labeled with ^(99m)Tc via cPN216 (% ID, % injected dose) in tumor,blood, liver, kidneys, bladder/urine, tail, intestine and spleen samplesfrom SKOV3 tumor bearing mice.

FIG. 12 is a graph of the biodistribution profile for Z02891 (SEQ. IDNo. 2) in SKOV3 tumor bearing mice showing the tumor:blood ratio.

FIGS. 13A and 13B are diagrams of the chemical structures forBoc-protected malimide-aminoxy (Mal-AO-Boc) and malimide-aminoxy(Mal-AO) linkers. 13A is the chemical structure for tert-butyl2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylamino)-2-oxoethoxycarbamate(Mal-AO-Boc) and 13B is the chemical structure for2-(aminooxy)-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)acetamidehydrochloride (Mal-AO.HCl).

FIG. 14A is the reverse phase HPLC chromatogram of Z00342 (SEQ. IDNo. 1) starting material and 14B is the reverse phase HPLC chromatogramof the purified Z00342 (SEQ. ID No. 1)-AO imaging agent composition,both analyzed at 280 nm.

FIG. 15 is the reverse phase HPLC gamma chromatogram for the crudereaction mixtures and purified final products of ¹⁸F-fluorobenzyl-Z00342(SEQ. ID No. 1) and ¹⁸F-fluorobenzyl-Z02891’ (SEQ. ID No. 2).

FIG. 16 is a graph of the biodistribution profile (% ID, % injecteddose) of the Z02891 (SEQ. ID No. 2) polypeptide labeled with ¹⁸F fromSKOV3-tumored animals.

FIG. 17 is a graph of the biodistribution profile of Z02891 (SEQ. ID No.2) polypeptide labeled with ¹⁸F (% ID, % injected dose) and thetumor:blood ratio from SKOV3-tumored animals.

FIG. 18 is bar graph of the biodistribution profile (% ID, % injecteddose) of ¹⁸F labeled Z00342 (SEQ. ID No. 1) and ¹⁸F labeled Z02891 (SEQ.ID No. 2) in blood, tumor, liver, kidneys, spleen and bone samples.

FIG. 19 is a diagram of the chemical structure of the Mal-NOTA linker.

FIG. 20A is a graph of the electrospray ionization time of flight massspectrum (ESI-TOF-MS), and 20B is a graph of the ESI-TOF-MS massdeconvolution result for Z00477 (SEQ. ID No. 3)-NOTA.

FIG. 21 is a graph of the reverse phase HPLC gamma trace for the crudereaction mixture of ⁶⁷Ga-labeled Z00477 (SEQ. ID No. 3)-NOTA after 1hour of reaction.

FIG. 22 is a graph of the reverse phase HPLC gamma trace for thepurified ⁶⁷Ga-labeled NOTA Z00477 (SEQ. ID No. 3)-NOTA polypeptide.

DETAILED DESCRIPTION

The imaging agents of the invention generally comprise an isolatedpolypeptide of SEQ. ID No. 1, SEQ. ID No. 2 or a conservative variantthereof, conjugated with ¹⁸F, ^(99m)Tc, ⁶⁷Ga or ⁶⁸Ga; and methods formaking and using the compositions. The isolated polypeptide bindsspecifically to HER2 or its variant thereof. In one or more embodiments,the sequence of the isolated polypeptide has at least 90% sequencesimilarity to any of SEQ. ID No. 1, SEQ. ID No. 2 or conservativevariant thereof.

The isolated polypeptide may comprise natural amino acids, syntheticamino acids, or amino acid mimetics that function in a manner similar tothe naturally occurring amino acids. Naturally occurring amino acids arethose encoded by the genetic code, as well as those amino acids that arelater modified, for example, hydroxyproline, γ-carboxyglutamate,O-phosphoserine, phosphothreonine, and phosphotyrosine.

The isolated polypeptides may be prepared using standard solid phasesynthesis techniques. Alternatively, the polypeptides may be preparedusing recombinant techniques. When the polypeptides are prepared usingrecombinant techniques, the DNA encoding the polypeptides orconservative variants thereof may be isolated. The DNA encoding thepolypeptides or conservative variants thereof may be inserted into acloning vector, introduced into a host cell (e.g., a eukaryotic cell, aplant cell, or a prokaryotic cell), and expressed using any artrecognized expression system.

The polypeptide may be substantially comprised of a single chiral formof amino acid residues. Thus, polypeptides of the invention may besubstantially comprised of either L-amino acids or D-amino acids;although a combination of L-amino acids and D-amino acids may also beemployed.

As the polypeptides provided herein are derived from the Z-domain ofprotein A, residues on the binding interface may be non-conservativelysubstituted or conservatively substituted while preserving bindingactivity. In some embodiments, the substituted residues may be derivedfrom any of the 20 naturally occurring amino acids or any analogthereof.

The polypeptides may be about 49 residues to about 130 residues inlength. The specific polypeptide sequences are listed in Table 1.

TABLE 1 Name Sequence Length Z00342 (SEQ. ID No. 1)GSSHHHHHHLQVDNKFNKEMRNA  72 YWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPKVDC Z02891 (SEQ. ID No. 2) AEAKYAKEMRNAYWEIALLPNLTN 61 QQKRAFIRKLYDDPSQSSELLSEAK KLNDSQAPKVDC Z00477 (SEQ. ID No. 3)VDNKFNKEMRNAYWEIALLPNLNV  61 AQKRAFIRSLYDDPSQSANLLAEAK KLNDAQAPKVDCZ00477-His6 (SEQ. ID No. 4) GSSHHHHHHLQVDNKFNKEMRNA  72(′His6′ disclosed as SEQ YWEIALLPNLNVAQKRAFIRSLYDD ID NO: 7)PSQSANLLAEAKKLNDAQAPKVDC (Z00477)₂ (SEQ. ID No. 5)GSSHHHHHHLQVDNKFNKEMRNA 130 YWEIALLPNLNVAQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPKVDN KFNKEMRNAYWEIALLPNLNVAQKRAFIRSLYDDPSQSANLLAEAKKLN DAQAPKVDC

Additional sequences may be added to the termini to impart selectedfunctionality. Thus, additional sequences may be appended to one or bothtermini to facilitate purification or isolation of the polypeptide,alone or coupled to a binding target (e.g., by appending a His tag tothe polypeptide).

The polypeptides listed in Table 1 may be conjugated with ¹⁸F via alinker; ^(99m)Tc via a diaminedioxime chelator, or with ⁶⁷Ga or ⁶⁸Ga viaa NOTA chelator. Table 2 provides the isoelectric point (pI), of thesepolypeptides.

TABLE 2 pI MW (kD) His6-Z00477 (SEQ. ID 8.31 8143.11 No. 4) (‘His6’disclosed as SEQ ID NO: 7) Z02891(SEQ. ID No. 2) 8.10 7029.96His6-Z00342 (‘His6’ 8.14 8318.27 disclosed as SEQ ID NO: 7)

In one or more embodiments, the isolated polypeptide, comprising SEQ. IDNo. 1, SEQ. ID No. 2 or a conservative variant thereof, may beconjugated with ¹⁸F. The ¹⁸F may be incorporated at a C terminus, at aN-terminus, or at an internal position of the isolated polypeptide.

In one or more embodiments, the ¹⁸F may be conjugated to the isolatedpolypeptide via a linker. The linker may comprise, an aminoxy group, anazido group, or an alkyne group. The aminoxy group of the linker may beattached with an aldehyde, such as a fluorine-substituted aldehyde. Anazide group of the linker may be attached with a fluorine substitutedalkyne. Similarly, an alkyne group of the linker may be attached with afluorine substituted azide. The linker may also comprise a thiolreactive group. The linker may comprise of a maleimido-aminoxy,maleimido-alkyne or maleimido-azide group. The ¹⁸F conjugatedpolypeptide may be prepared by: (i) providing the isolated polypeptidecomprising SEQ.ID No. 1, SEQ.ID No. 2, or a conservative variantthereof; (ii) reacting the polypeptide with a linker, wherein the linkercomprises an aminoxy group, an azido group, or an alkyne group, to forma linker conjugated polypeptide; and reacting the linker with an ¹⁸Fmoiety.

In another embodiment, the method may comprise: (i) providing anisolated polypeptide comprising SEQ. ID No. 1, SEQ. ID No. 2, or aconservative variant thereof; (ii) providing a linker; (iii) reactingthe linker with the ¹⁸F moiety to form a ¹⁸F labeled linker; and (iv)reacting the ¹⁸F labeled linker with the isolated polypeptide of SEQ. IDNo 1, SEQ ID no 2, or a conservative variant thereof, to form a linkerconjugated polypeptide.

Using the above-described examples, fluorine or radiofluorine atom(s),such as ¹⁸F, may be introduced onto the polypeptides. Afluorine-substituted polypeptide results when a fluorine-substitutedaldehyde is reacted with the aminoxy group of the linker conjugatedpolypeptide. Similarly, a fluorine substituted polypeptide results, whena fluorine substituted azide or alkyne group is reacted with therespective alkyne or azide group of the linker conjugated polypeptide. Aradiofluorine-labeled polypeptide or imaging agent composition results,when a radiofluorine-substituted aldehyde, azide or alkyne is reactedwith the respective aminoxy, alkyne or azide group of the linkerconjugated polypeptide. Further, each of the aldehydes, azides oralkynes may have a radiofluorine (¹⁸F) substituent, to prepareradiofluorine-labeled imaging agent compositions. The methods forintroducing fluorine onto the polypeptide may also be used to prepare afluorinated imaging agent composition of any length. Thus, in someembodiments the polypeptide of the imaging agent composition maycomprise, for example, 40 to 130 amino acid residues.

The chemistry for the synthesizing linker-conjugated polypeptide of theimaging agents is facile, and the one step reaction of the methods aremore efficient than previously known methods and result in higheryields. The methods are easier to carry out, faster and are performedunder milder, more user friendly, conditions. For example, the methodfor labeling a polypeptide with ¹⁸F conjugated with a linker (e.g.,¹⁸F-fluorobenzaldehyde) is simpler than the procedures known in the art.¹⁸F conjugated-linker is prepared in one step by the direct nucleophilicincorporation of ¹⁸F onto the trimethylanilinium precursor. ¹⁸F-linker(i.e., ¹⁸F-FBA) is then conjugated to the polypeptide, such as anaffibody. The preparation of the linker is also easier than previouslyknown methods in the art. Moreover, radiolabeled aminoxy basedlinker-conjugated polypeptides, and the cPn family of chelatorconjugated polypeptides (e.g., affibody), show significantly betterbiodistribution and better tumor uptake, as well as better clearancewith less liver uptake.

The fluorine-labeled compositions are highly desired materials indiagnostic applications. ¹⁸F labeled imaging agent compositions may bevisualized using established imaging techniques such as PET.

In another embodiment, the polypeptide may be conjugated with ^(99m)Tcvia a diamindioxime chelator of formula (1).

wherein R^(/), R^(//), R^(///), R^(////) is independently H or C₁₋₁₀alkyl, C₃₋₁₀ alkylary, C₂₋₁₀ alkoxyalkyl, C₁₋₁₀ hydroxyalkyl, C₁₋₁₀alkylamine, C₁₋₁₀ fluoroalkyl, or 2 or more R groups, together with theatoms to which they are attached to form a carbocyclic, heterocyclic,saturated or unsaturated ring, wherein R may be H, C₁₋₁₀ alkyl, C₃₋₁₀alkylary, C₂₋₁₀ alkoxyalkyl, C₁₋₁₀ hydroxyalkyl, C₁₋₁₀ alkylamine, orC₁₋₁₀ fluoroalkyl. In one embodiment, n may vary from 0-5. Examples ofmethods for preparing diaminedioxime chelators are described in PCTApplication, International Publication No. WO2004080492(A1) entitled“Methods of radio fluorination of biologically active vector”, and PCTApplication, International Publication No. WO2006067376(A2) entitled“Radio labelled conjugates of RGD-containing peptides and methods fortheir preparation via click-chemistry”, which are incorporated herein byreferences.

The ^(99m)Tc may be conjugated to the isolated polypeptide via thediamindioxime at the N-terminus of the isolated polypeptide. Thechelator may be a bifunctional compound. In one embodiment, thebifunctional compound may be Mal-cPN216. The Mal-cPN216 comprises athiol-reactive maleimide group for conjugation to a terminal cysteine ofthe polypeptide of SEQ ID No. 1 or SEQ ID No 2 and a bis-amineoximegroup (diamindioxime chelator) for chelating with ^(99m)Tc. TheMal-cPN216 may have a formula (2).

The diamindioxime chelator conjugated peptide may be prepared by (i)providing an isolated polypeptide comprising SEQ.ID No. 1, SEQ. ID No. 2or a conservative variant thereof, (ii) reacting a diamindioximechelator with the polypeptide to form the diamindioxime conjugatedpolypeptide. The diamindioxime chelator may be further conjugated with^(99m)Tc.

In one or more embodiments, the polypeptide may be conjugated with ⁶⁷Ga,or ⁶⁸Ga via NOTA (1,4,7-triazacyclononane-N,N′,N″-triacetic acid)chelator. The NOTA conjugated polypeptide may be prepared by (i)providing an isolated polypeptide comprising SEQ.ID No. 1, SEQ. ID No. 2or a conservative variant thereof, (ii) reacting a NOTA chelator withthe polypeptide to form the NOTA conjugated polypeptide. The NOTAchelator may be further conjugated with ⁶⁷Ga or ⁶⁸Ga.

In one embodiment, the Ga, specifically ⁶⁷Ga, may be conjugated to theisolated polypeptide via NOTA chelator. The NOTA chelator may befunctionalized with a maleimido group, as described in formula (3).

The invention also comprises methods of imaging at least a portion of asubject. In one embodiment, the method comprises administering theimaging agent composition to the subject and imaging the subject. Thesubject may be imaged, for example, with a diagnostic device.

The method may further comprise the steps of monitoring the delivery ofthe composition to the subject and diagnosing the subject with aHER2-associated disease condition (e.g., breast cancer). In oneembodiment, the subject may be a mammal, for example, a human. Inanother embodiment, the subject may comprise cells or tissues. Thetissues may be used in biopsy. The diagnostic device may employ animaging method chosen from magnetic resonance imaging, optical imaging,optical coherence tomography, X-ray, computed tomography, positronemission tomography, or combinations thereof.

The imaging agent compositions may be administered to humans and otheranimals parenterally. Pharmaceutical compositions of this invention forparenteral injection comprise pharmaceutically-acceptable sterileaqueous or nonaqueous solutions, dispersions, suspensions or emulsionsas well as sterile powders for reconstitution into sterile injectablesolutions or dispersions just prior to use. Examples of suitable aqueousand nonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), and suitable mixtures thereof, vegetable oils(such as olive oil), and injectable organic esters such as ethyl oleate.Proper fluidity can be maintained, for example, by using coatingmaterials such as lecithin, by adjusting the particle size indispersions, and by using surfactants.

These imaging agent compositions may also contain adjuvant such aspreservatives, wetting agents, emulsifying agents, and dispersingagents. Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents such as sugars, sodium chloride,and the like. Prolonged absorption of the injectable pharmaceutical formmay be brought about by the inclusion of agents, which delay absorptionsuch as aluminum monostearate and gelatin.

The imaging agent compositions may be dispersed in physiologicallyacceptable carrier to minimize potential toxicity. Thus, the imagingagents may be dispersed in a biocompatible solution with a pH of about 6to about 8. In some embodiments, the agent is dispersed in abiocompatible solution with a pH of about 7 to about 7.4. In otherembodiments, the agent is dispersed in a biocompatible solution with apH of about 7.4.

The imaging agent compositions may be combined with other additives thatare commonly used in the pharmaceutical industry to suspend or dissolvethe compounds in an aqueous medium, and then the suspension or solutionmay be sterilized by techniques known in the art. The imaging agentcomposition may be administered in a variety of forms and adapted to thechosen route of administration. For example, the agents may beadministered topically (i.e., via tissue or mucus membranes),intravenously, intramuscularly, intradermally, or subcutaneously. Formssuitable for injection include sterile aqueous solutions or dispersionsand sterile powders for the preparation of sterile injectable solutions,dispersions, liposomal, or emulsion formulations. Forms suitable forinhalation use include agents such as those dispersed in an aerosol.Forms suitable for topical administration include creams, lotions,ointments, and the like.

The imaging agent compositions may be concentrated to convenientlydeliver a preferred amount of the agents to a subject and packaged in acontainer in the desired form. The agent may be dispensed in a containerin which it is dispersed in a physiologically acceptable solution thatconveniently facilitates administering the agent in concentrationsbetween 0.1 mg and 50 mg of the agent per kg body weight of the subject.

In one or more embodiments, the target tissue may be imaged about fourhours after administering the agents. In alternative embodiments, thetarget tissue may be imaged about 24 hours after administering theagents to the subject.

Examples

The following examples are provided for illustration only and should notbe construed as limiting the invention.

A panel of tumorigenic cell lines with a reasonable probability ofexpressing HER2 was selected based on available literature (Bruskin, et.al. Nucl. Med. Biol. 2004: 31: 205; Tran, et. al. Imaging agentcomposition Chem. 2007: 18: 1956), as described in Table 3.

TABLE 3 Cell line Species Type Purpose SKOV3 Human Ovarian carcinomaCandidate SKBR3 Human Breast carcinoma Candidate C6 Rat Glioma control

All cell lines were obtained from the American Type Culture Collection(ATCC) and cultured as recommended. Cells were cultured to >90%confluence prior to use. Flow cytometry (Beckman Coulter Cytomics FC500MPL) was carried out on the cell lines listed in table 4 using anti-Her2primary antibodies (R&D Systems, PN MAB1129) and the Dako QIFIKIT (PNK0078) for quantitative analysis of indirect immunofluorescencestaining. Calibration beads with 5 different populations bearingdifferent numbers of Mab molecules were used in conjunction with thecell lines to determine number of surface receptors per cell. In allcases, appropriate isotype controls were obtained from the correspondingvendors.

Adherent cells were released from their flasks using cell dissociationbuffer (PBS+10 mM EDTA) rather than trypsin to avoid proteolysis of cellsurface receptors. Cells were washed twice in PBS and resuspended inice-cold FC buffer (PBS+0.5% BSA w/v) to a concentration of 5-10×106cells/ml. 100 μL aliquots of cells were mixed with 5 μg of primaryantibody and incubated, on ice, for 45 minutes. Cells were then washedwith 1 ml of ice cold flow cytometry (FC) buffer (PBS with 2% bovineserum albumin), centrifuged at 300×g for 5 min, and resuspended in 0.5μL of FC buffer. 100 μL of 1:50 dilution with PBS of the secondaryantibody fragment (F(ab)₂ FITC-conjugated goat anti-mouseImmunoglobulins) was added and incubated, on ice and in the dark, for 45minutes. Cells were then washed twice with 1 mL of ice cold FC buffer,centrifuged at 300×g for 5 min, and resuspended in 500 μL of FC buffer.All stained cells were passed through a 100-micron filter prior to flowcytometry to prevent clogs of the flow cell.

Flow cytometry was carried out on a Beckman Coulter Cytomics FC500 MPL.A minimum of 5×10⁴ events was collected for each tube. All analyses weresingle color, with detection of FITC in FL1. Forward scatter (FS) andside scatter (SS) data demonstrated that all cell populations weretightly grouped.

Flow cytometry was used to evaluate the cells for their HER2 expressionin vitro (FIGS. 2A, 2B, and 2C) with SKOV3 cells showing the highestlevel of HER2 expression (FIG. 3). The results in FIG. 3 werereproducible (n=3).

The highest expressing cell line was SKOV3. These cells were injectedinto 6-12 week old immuno-compromised mice and allowed to grow tumors.Tumor growth curves and success rates were dependent on the number ofcells inoculated. Optimal tumor growth was obtained with three to fourmillion cells/mouse

In vivo studies were carried out with female CD-1 nude mice (CharlesRiver Labs, Hopkinton, Mass.) with an age range between 6 and 15 weeks.Mice were housed in a ventilated rack with food and water ad libitum anda standard 12 hour day-night lighting cycle. For xenografts, animalswere injected with 100 μl of cells in PBS. Cells were implantedsubcutaneously in the right hindquarter. Implantation was performedunder isoflurane anesthesia. For SKOV3, between 3×10⁶ to 4×10⁶ cellswere implanted in each mouse. Under these conditions, useable tumors(100 to 300 μg) were obtained in 3 to 4 weeks in greater than 80% ofanimals injected.

Tumors were collected from mice by dissection, and whole tumors werestored at −20° C. until processing. Tumors were ground on ice in 1 ml ofRIPA buffer supplemented with a protease inhibitor cocktail (Santa CruzBiotech, Santa Cruz, Calif. #24948) in a Dounce homogenizer. Homogenateswere then incubated on ice for 30 minutes, then centrifuged at 10,000×Gfor 10 minutes in a refrigerated centrifuge. Supernatants were collectedand stored on ice or at 4° C. until further processing. Proteinconcentrations in lysates were determined using a BCA protein assay kit(Pierce Biotechnology 23225). Lysates were diluted to a standardconcentration to yield 20 μg of protein/well in the microtiter plate.ELISA's were run with a commercially available human HER2 kit (R&DSystems, DYC1129) according to the manufacturer's instructions. Eachsample was run in triplicate, and data are reported as pg HER2/μg totalprotein, errors are reported as standard deviations.

Target expression in vivo was measured by ELISA. Excised tumors werehomogenized and analyzed for HER2 using a commercially available matchedpair kit (R&D systems, DYC1129, Minneapolis, Minn.). The results, inFIG. 3, show that the SKOV3 cell line grows a high-expressing tumor.ELISA controls were cell-culture lysates of the negative control linesused for flow cytometry. These results indicate that tumor xenografts ofSKOV3 are appropriate for the in vivo study of molecules targeting humanHER2.

All polypeptides were received from Affibody AB in Sweden. Thepolypeptides are referred to by their numeric internal developmentcodes, which are prefixed with “Z”. Table 1 details the polypeptidesdescribed herein. The polypeptides include polypeptide Z00342 (SEQ. IDNo. 1); polypeptide Z02891 (SEQ. ID No. 2); polypeptide Z00477 (SEQ. IDNo. 3 and 4), and dimer of Z00477, i.e., (Z00477)₂ (SEQ. ID No. 5).

Binding interactions between the polypeptides and the HER2/neu antigenwere measured in vitro using surface plasmon resonance (SPR) detectionon a Biacore™ 3000 instrument (GE Healthcare, Piscataway, N.J.). Theextracellular domain of the Her2/neu antigen was obtained as a conjugatewith the Fc region of human IgG (Fc-Her2) from R&D Systems (Minneapolis,Minn.) and covalently attached to a CM-5 dextran-functionalized sensorchip (GE Healthcare, Piscataway, N.J.) pre-equilibrated with HBS-EPbuffer (0.01M HEPES pH 7.4, 0.15M NaCl, 3 mM EDTA, 0.005% v/v surfactantP20) at 10 μL/min and subsequently activated with EDC and NHS. TheFc-HER2 (5 μg/ml) in 10 mM sodium acetate (pH 5.5) was injected onto theactivated sensor chip until the desired immobilization level (˜3000Resonance Units) was achieved (2 min). Residual activated groups on thesensor chip were blocked by injection of ethanolamine (1 M, pH 8.5). Anynon-covalently bound conjugate was removed by repeated (5×) washing with2.5 M NaCl, 50 mM NaOH. A second flow cell on the same sensor chip wastreated identically, except with no Fc-HER2 immobilization, in order toserve as a control surface for refractive index changes and non-specificbinding interactions with the sensor chip. Prior to the kinetic study,binding of the target analyte was tested on both surfaces and a surfacestability experiment was performed to ensure adequate removal of thebound analyte and regeneration of the sensor chip following treatmentwith 2.5 M NaCl, 50 mM NaOH. SPR sensorgrams were analyzed using theBIAevaluation software (GE Healthcare, Piscataway, N.J.). The robustnessof the kinetic model was determined by evaluation of the residuals andstandard error for each of the calculated kinetic parameters, the“goodness of the fit” (χ²<10), and a direct comparison of the modeledsensorgrams to the experimental data. SPR measurements were collected ateight analyte concentrations (0-100 nM protein) and the resultingsensorgrams were fitted to a 1:1 Langmuir binding model.

FIG. 1 shows example surface plasmon resonance (SPR) data obtained forZ00477 (SEQ. ID No. 3) and (Z00477)₂ (SEQ. ID No. 5) when run on humanHER2-functionalized surfaces. This relationship holds true for allpolypeptides for which the affinities are known (Table 2), in which thevalues for the dimer Z(477)2 (SEQ. ID No. 5) are estimates based onavidity affect.

Labeling of His6 (SEQ ID NO: 7)-tagged Polypeptides with thefac-[^(99m)Tc(CO)₃]⁺ core was accomplished using modifications to apreviously published procedure (Waibel, R.; et al., A. Nat. Biotechnol.1999, 17, 897.). Briefly, Na[^(99m)TcO₄] in saline (4 mCi, 2 mL) wasadded to an Isolink® boranocarbonate kit (Alberto, R. et al, J. Am.Chem. Soc. 2001, 123, 3135.). The resulting solution was heated to 95°C. for 15-20 minutes, to give fac-[^(99m)Tc(CO)₃(H₂O)₃]⁺. A portion (2mCi, 1 mL) of the solution was removed and neutralized to pH ˜7 with 1 NHCl. A 325 μL aliquot was removed and added to a solution of theHis6-Polypeptide (SEQ ID NO: 7) (40 μg). The resulting solution washeated in a water bath at 35-37° C. for 40 minutes. Typicalradiochemical yields ranged from 80-95% (determined by ITLC-SG, Biodex,0.9% NaCl). The crude reaction products were chromatographed on a NAP-5column (GE Healthcare, 10 mM PBS) to give products of >99% radiochemicalpurity. Typical specific activities obtained were 3-4 μCi/μg. Theresulting solution was then diluted with 10 mM PBS to give the properconcentration for subsequent biodistribution studies.

HPLC was carried out on an Agilent 1100 series HPLC equipped with aGrace-Vydac Peptide/Protein C4 (4.6×250 mm) column and a Raytest GABIradioactivity detector. Solvent A was 95:5 water:MeCN with 0.1% TFA, andsolvent B was 5:95 water:MeCN with 0.1% TFA. The gradient was as follows(all changes linear; time/% B): 0/0, 4/20, 16/60, 20/100, 25/100, 26/0,31/0.

Each polypeptide was labeled with the tricarbonyltechnetium core in highyield (>90%) before purification. Purification by NAP-5 chromatographygave samples of ^(99m)Tc-labeled Polypeptides in >99% radiochemicalpurity (Table 4)

TABLE 4 Crude Isolated yield NAP-5 yield (decay corr.) RCP Compound (%)(%) (%) Z00477 (SEQ. ID No. 3) 56.9 24.7 (26.9) 99.5

Representative HPLC chromatograms of NAP-5 purified radiolabeledpolypeptides are shown in FIG. 4. The retention time of the radiolabeledspecies was virtually unchanged from the corresponding unlabeledpolypeptide's retention time in a 220 nm UV chromatogram (except for thetime difference due to the physical separation of the UV and gammadetectors; data not shown).

Animal Models used to study ⁹⁹Tc(CO)₃(His₆)-Polypeptides (‘His₆’disclosed as SEQ ID NO: 7)

In vivo studies were carried out with female CD-1 nude mice (CharlesRiver Labs, Hopkinton, Mass.) with an age range between 6 and 15 weeks.Mice were housed in a ventilated rack with food and water ad libitum anda standard 12 hour day-night lighting cycle. For xenografts, animalswere injected with 100 μl of cells in PBS. Cells were implantedsubcutaneously in the right hindquarter. Implantation was performedunder isoflurane anesthesia. For SKOV3, between 3×10⁶ to 4×10⁶ cellswere implanted in each mouse. Under these conditions, useable tumors(100 to 300 μs) were obtained in 3 to 4 weeks in greater than 80% ofanimals injected.

Biodistribution

Mice were given tail-vein injections of ˜1 μg of ^(99m)Tc-labeledpolypeptides (˜3 μCi/1 μg). Mice were placed in filter-paper lined cagesuntil euthanasia. Three mice were euthanized at each timepoint andtissues of interest dissected and counted on a Perkin Elmer WallacWizard 1480 Gamma Counter. Data were collected for blood, kidney, liver,spleen, and injection site (tail). Urine from cages was pooled with thebladder and also counted. The remaining tissues were counted and the sumof all tissues plus urine for each animal was summed to provide thetotal injected dose. The % injected dose for each organ was determinedbased on this total, and organs were weighed for determination of the %injected dose per gram, (% ID/g). Data is reported as mean value for allthree mice in the timepoint with error bars representing the standarddeviation of the group.

The ^(99m)Tc labeled Z00477 (SEQ. ID No. 4) polypeptide was injectedinto SKOV3 mice. FIG. 6 shows the tumor and blood curves for theseexperiments. The Z00477 (SEQ. ID No. 4) polypeptide shows good tumoruptake in target-expressing SKOV3 tumors, with a maximal value ofapproximately 3% of the injected dose per gram of tissue at 30 minutespost-injection (PI), and a peak tumor:blood ratio of more than 8 at 240minutes PI.

Polypeptides exhibit a monoexponential clearance from the blood withhalf-lives of less than two minutes. This clearance is primarilymediated by the liver and kidneys. Polypeptide uptake in the spleen wasmoderate, and moderate to high uptake in the liver is observed, asdescribed in Table 5.

TABLE 5 Z00477 (SEQ. ID No. 3) His6 (SEQ ID NO: 7)tagged uptake (% ID/g)in SKOV3 tumor bearing mice 5 Minutes 30 Minutes 120 Minutes 240 MinutesBlood 7.30 ± 0.32 1.47 ± 0.16 0.56 ± 0.03 0.43 ± 0.03 (n = 3) (n = 3) (n= 3) (n = 3) Tumor 1.57 ± 0.62 3.06 ± 0.17 3.40 ± 0.87 3.60 ± 1.15 (n =3) (n = 3) (n = 3) (n = 3) Liver 29.07 ± 0.70 32.19 ± 6.50 39.57 ± 6.2935.17 ± 3.48 (n = 3) (n = 3) (n = 3) (n = 3) Kidney 54.83 ± 9.29 85.89+10.00 97.99 ± 10.45 92.54 ± 7.36 (n = 3) (n = 3) (n = 3) (n 3) Spleen5.57 ± 2.39 3.76 ± 0.23 4.65 ± 2.21 5.36 ± 0.80 (n = 3) (n = 3) (n = 3)(n = 3)

Bivalent polypeptides exhibit higher affinity than the correspondingmonomers, presumably due to the avidity effect. Their larger size,however, may hinder tumor penetration. For the HER2 polypeptides,bivalent forms of each the four high affinity polypeptides wereavailable. The Z00477 (SEQ. ID No. 3) dimer, (Z00477)₂ (SEQ. ID No. 5),was radiolabeled and used for a four-hour biodistribution experiment inSKOV3-tumored mice.

The monovalent and bivalent polypeptides otherwise exhibit similarbiodistribution characteristics, and blood half-lives are observed forboth in the one to two minute range. The results clearly indicate thatboth monomeric and divalent polypeptides can be targeted to HER2 invivo.

To introduce the ^(99m)Tc chelator cPN216 (FIG. 7), a bifunctionalcompound Mal-cPN216 was synthesized comprising of a thiol-reactivemaleimide group for conjugation to a terminal cysteine of a polypeptideand an amine oxime group for chelating ^(99m)Tc.

cPN216-amine was obtained from GE Healthcare. N-ß-maleimidopropionicacid was purchased from Pierce Technologies (Rockford, Ill.).N-methylmorpholine, (benzotriazol-1-yloxy) tripyrrolidinophosphoniumhexafluorophosphate (PyBoP), dithiothreitol (DTT), ammonium bicarbonate,and anhydrous DMF were purchased from Aldrich (Milwaukee, Wis.). PBSbuffer (1×, pH 7.4) was obtained from Invitrogen (Carlsbad, Calif.).HPLC-grade acetonitrile (CH₃CN), HPLC-grade trifluoroacetic acid (TFA),and Millipore 18 mΩ, water were used for HPLC purifications.

To an ice-cooled solution of N-β-maleimidopropionic acid (108 mg, 0.64mmol), cPN216-amine (200 mg, 0.58 mmol), and PyBoP (333 mg, 0.64 mmol)in anhydrous DMF at 0° C. was added 0.4 M of N-methylmorpholine in DMF(128 μL, 1.16 mmol). The ice bath was removed after 2 hrs, and themixture was stirred at room temperature overnight before being subjectedto HPLC purification. The product was obtained as a white powder (230mg, 80% yield). 1H-NMR (400 MHz, DMSO-d6): δ 1.35 (m, 2H), 1.43 (s,12H), 1.56 (m, 5H), 1.85 (s, 6H), 2.33 (dd, J1=8 Hz, J2=4 Hz, 2H), 2.78(m, 4H), 3.04 (m, 2H), 3.61 (dd, J1=8 Hz, J2=4 Hz, 2H), 7.02 (s, 2H),8.02 (s, 1H), 8.68 (s, 4H), 11.26 (s, 2H); m/z=495.2 for [M+H]⁺(C24H43N6O5, Calculated MW=495.3).

The polypeptide was dissolved with freshly degassed PBS buffer (1×, pH7.4) at a concentration of approximately 1 mg/mL. The disulfide linkagein the polypeptide was reduced by the addition of DTT solution infreshly degassed PBS buffer (1×, pH 7.4). The final concentration of DTTwas 20 mM. The reaction mixture was vortexed for 2 hours and passedthrough a Zeba desalt spin column (Pierce Technologies) pre-equilibratedwith degassed PBS buffer (1×, pH 7.4) to remove excess of DTT reagent.The eluted reduced polypeptide molecule was collected, and thebifunctional compound Mal-cPN216 was added (20 equivalents perequivalent of the polypeptide) as a solution in DMSO, and the mixturewas vortexed at room temperature for 3 hours and frozen withliquid-nitrogen. The reaction mixture was stored overnight before beingsubject to Reverse phase HPLC purification (FIGS. 8A and 8B).

The HPLC purification was performed on a MiCHROM Magic C18AQ 5μ 200Acolumn (MiChrom Bioresources, Auburn, Calif.). Solvent A: H₂O (with 0.1%formic acid), Solvent B: CH₃CN (with 0.1% formic acid). Gradient: 5-100%B over 30 mins.

The fractions containing desired product were combined and neutralizedwith 100 mM ammonium bicarbonate solution, and the solvents were removedby lyophilization to give the desired imaging agent composition as awhite solid (yield 41%).

LC-MS analysis of the purified product confirmed the presence of thedesired product, and the MW suggested that only one cPN216 label wasadded to polypeptide constructs (Z00477 (SEQ. ID No. 3)-cPN216:calculated MW: 7429 Da, found: 7429 Da; Z02891 (SEQ. ID No. 2)-cPN216calculated MW: 7524 Da, found: 7524 Da).

To a 20 mL vial was added 10.00 mL of distilled, deionized water.Nitrogen gas was bubbled through this solution for approximately 30minutes prior to addition of the NaHCO₃(450 mg, 5.36×10⁻³ mol), Na₂CO₃(60 mg, 5.66×10⁻⁴ mol) and sodium para-aminobenzoate (20 mg, 1.26×10⁻⁴mol). All reagents were weighed independently and added to the vialcontaining water. Tin chloride (1.6 mg, 7.09×10⁻⁶ mol) and MDP (2.5 mg,1.42×10⁻⁵ mol) were weighed together into a 1 dram vial and subsequentlytransferred (with 1 subsequent wash) by rapid suspension inapproximately 1 mL of the carbonate buffer mixture. 10 μL aliquots wereremoved and transferred under a stream of nitrogen to silanized vials,immediately frozen and maintained in a liquid nitrogen bath untillyophilization. Each vial was partially capped with rubber septa andplaced in a tray lyophilizer overnight. Vials were sealed under vacuum,removed from the lyophilizer, crimp-sealed with aluminum caps,re-pressurized with anhydrous nitrogen and stored in a freezer untilfuture use.

Synthesis of the radiolabeled polypeptide was performed using a one-stepkit formulation produced in house (Chelakit A+) containing a lyophilizedmixture of stannous chloride as a reducing agent for technetium,methylene diphosphonic acid, p-aminobenzoate as a free-radical scavengerand sodium bicarbonate/sodium carbonate (pH 9.2) as buffer. In rapidsuccession, 20 μL of a 2 μg/μL solution of polypeptide in saline wasadded to the Chelakit, followed immediately by Na^(99m)TcO₄ (0.8 mCi,29.6 MBq) in 0.080 mL of saline (0.15M NaCl) obtained from CardinalHealth (Albany, N.Y.). The mixture was agitated once and allowed to sitat ambient temperature for 20 min. Upon completion, the cruderadiochemical yield was determined by ITLC (Table 6 below according toITLC-SG, Biodex, 0.9% NaCl).

TABLE 6 RCY (%) Crude purified RCP decaycorrected/ Compound RCP (%) (%)(uncorrected) Z00477 (SEQ. ID No. 3) 49.2 98.6 53.9 (13.1) Z02891 (SEQ.ID No. 2) 71.6 97.5 46.9(43.8)

The reaction volume was increased to 0.45 mL with 0.35 mL of 150 mMsterile NaCl, and the final product purified by size exclusionchromatography (NAPS, GE Healthcare, charged with 10 mM PBS). The crudereaction mixture was loaded onto the NAPS column, allowed to enter thegel bed and the final purified product isolated after elution with 0.8mL of 10 mL PBS. Final activity was assayed in a standard dosecalibrator (CRC-15R, Capintec, Ramsey, N.J.). Radiochemical yield (Table6) and purity were determined by ITLC (>98.5%), C4 RP-HPLC (FIG. 9) andSEC-HPLC analysis. The final product (10-15 μCi/μg, 0.2-0.5 μCi/μL (0.37MBq/μg, 7.4 MBq/mL)) was used immediately for biodistribution studies.

The HPLC conditions used for this experiment were as follows: C4 RP-HPLCmethod 1: Solvent A: 95/5 H₂O/CH₃CN (with 0.05% TFA), Solvent B: 95/5CH₃CN/ddH₂O (distilled, deionized water) with 0.05% TFA. Gradientelution: 0 min. 0% B, 4 min. 20% B, 16 min. 60% B, 20 min. 100% B, 25min. 100% B, 26 min. 0% B, 31 min. 0% B.

C4 RP-HPLC method 2: Solvent A: 0.06% NH₃ in water, Solvent B: CH₃CN.Gradient elution: 0 min. 0% B, 4 min. 20% B, 16 min. 60% B, 20 min. 100%B, 25 min. 100% B, 26 min. 0% B, 31 min. 0% B.

RP-HPLC analysis performed on an HP Agilent 1100 with a G1311A QuatPump,G1313A autoinjector with 100 μL syringe and 2.0 mL seat capillary, GraceVydac—protein C4 column (S/N E050929-2-1, 4.6 mm×150 mm), G1316A columnheater, G1315A DAD and Ramon Star-GABI gamma-detector.

SEC HPLC: Solvent: 1× (10 mM) PBS (Gibco, Invitrogen, pH 7.4 containingCaCl₂) and MgCl₂). Isocratic elution for 30 min. Analysis performed ona: Perkin Elmer SEC-4 Solvent Environmental control, Series 410 LC pump,ISS 200 Advanced LC sample processor and Series 200 Diode ArrayDetector. A Raytest GABI with Socket 8103 0111 pinhole (0.7 mm innerdiameter with 250 μL volume) flow cell gamma detector was interfacedthrough a Perkin Elmer NCI 900 Network Chromatography Interface. Thecolumn used was a Superdex 75 10/300 GL High Performance SEC column (GEHealthcare. code: 17-5174-01, ID no. 0639059).

The operating pH of the Chelakits used to incorporate ^(99m)Tc into thecPN216 chelate (pH=9.2) nearly matched the calculated pI of the Z00477(SEQ. ID No. 3) polypeptide. Labeling under these conditions weredetermined to cause aggregation in the final product (FIGS. 5A and 5B).Aggregation was confirmed by size exclusion HPLC and through theincreased blood residence time and liver uptake observed in thebiodistribution studies. By altering the isoelectric point of thepolypeptide, ^(99m)Tc was successfully incorporated onto the Z02891(SEQ. ID No. 2) construct. Size exclusion HPLC confirmed the presence ofa species with the appropriate molecular weight and biodistributionstudies showed uptake of the tracer into the tumor xenografts.

In vivo studies were carried out with female CD-1 nude mice (CharlesRiver Labs, Hopkinton, Mass.) with an age range between 6 and 15 weeks.Mice were housed in a ventilated rack with food and water ad libitum anda standard 12 hours day-night lighting cycle. For xenografts, animalswere injected with 100 μl of cells in PBS. Cells were implantedsubcutaneously in the right hindquarter. Implantation was performedunder isoflurane anesthesia. For SKOV3, between 3×10⁶ to 4×10⁶ cellswere implanted in each mouse. Under these conditions, useable tumors(100 to 300 μs) were obtained in 3 to 4 weeks in greater than 80% ofanimals injected.

Mice were given tail-vein injections of ˜1 μg of ^(99m)Tc-labeledpolypeptides (˜10 μCi/1 μg). Mice were placed in filter-paper linedcages until euthanasia. Three mice were euthanized at each timepoint andtissues of interest dissected and counted on a Perkin Elmer WallacWizard 1480 Gamma Counter. Data were collected for blood, kidney, liver,spleen, and injection site (tail). Urine from cages was pooled with thebladder and also counted. The remaining tissues were counted and the sumof all tissues plus urine for each animal was summed to provide thetotal injected dose. The % injected dose for each organ was determinedbased on this total, and organs were weighed for determination of the %injected dose per gram, (% ID/g). Data is reported as mean value for allfour to five mice in the time point with error bars representing thestandard deviation of the group. Four time points were taken over fourhours (5, 30, 120, and 240 minutes post-injection).

The Z02891 (SEQ. ID No. 2)-cPN216-^(99m)TC polypeptide shows strongtumor uptake in target-expressing SKOV3 tumors, with a value of7.11±1.69% (n=5) of the injected dose per gram of tissue at 30 minutespost-injection (PI), which remains fairly constant over the time-courseof the study up to 240 min PI. Tumor:blood ratios were 2, 5, and 5 at30, 120, and 240 min post injection, respectively. FIGS. 10, 11 and 12show the tumor, blood and tumor:blood curves for these experiments.

The Polypeptides exhibit a monoexponential clearance from the blood withhalf-lives of less than two minutes. This clearance is primarilymediated by the kidneys, with 10.58±2.96 (n=5) ID/organ at 240 minpost-injection PI. Activity is secreted primarily in the urine.Polypeptide uptake in the spleen was moderate to high due to possibleaggregation, and moderate uptake in the liver is observed, e.g., 12%ID/organ (equivalent in value in mice to % ID/g) over the course of thestudy.

Biodistribution Results for Z02891 (SEQ. ID No. 2)-cPN216-^(99m)Tc

TABLE 7 Z02891 (SEQ. ID No. 2) cPN216 uptake (% ID/g) in SKOV3 tumorbearing mice 5 Minutes 30 Minutes 120 Minutes 240 Minutes Blood 8.69 ±0.99 3.32 ± 0.48 1.33 ± 0.05 1.05 ± 0.09 (n = 5) (n = 5) (n = 5) (n = 5)Tumor 3.19 ± 1.78 7.11 ± 1.69 7.18 ± 3.33 5.07 ± 3.47 (n = 5) (n = 4) (n= 5) (n = 5) Liver 9.87 ± 0.81 11.07 ± 1.06 8.33 ± 0.50 9.38 ± 0.69 (n =5) (n = 5) (n = 5) (n = 5) Kidney 67.61 ± 9.24 74.15 ± 4.17 37.14 ± 3.4829.67 ± 10.87 (n = 5) (n = 5) (n = 5) (n = 5) Spleen 7.07 ± 1.84 4.51 ±1.25 3.91 ± 0.44 2.85 ± 0.62 (n = 5) (n = 5) (n = 5) (n = 5)

Z00477 (SEQ. ID. NO. 4), Z00342 (SEQ. ID No. 1) and Z02891 (SEQ. ID No.2)-cysteine polypeptides were functionalized with an aminoxy group viaan engineered C-terminal cysteine. The purity of the polypeptidemolecules provided was determined to be >95% by High Performance LiquidChromatography (HPLC).

To incorporate ¹⁸F into the Polypeptide molecules, a bifunctional linkerMal-AO was synthesized comprising of two orthogonal groups: athiol-reactive maleimide group for conjugation to the engineeredcysteine and an aldehyde-reactive aminoxy group (FIGS. 13A and 13B).This linker was prepared by reacting N-(2-aminoethyl) malemide with2-(tert-butoxycarbonylaminooxy) acetic acid using1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC)-mediated couplingconditions yielding the Boc-protected form of the linker. The Bocprotecting group was then de-protected by acid cleavage to give thefinal Mal-AO product in quantitative yield. The final product was useddirectly without further purification.

Dichloromethane, 2-(tert-butoxycarbonylaminooxy) acetic acid,triethylamine, N-(2-aminoethyl)maleimide trifluoroacetic acid (TFA)salt, N-hydroxybenzotriazole hydrate (HOBT),1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC), dithiothriotol(DTT), and all other standard synthesis reagents were purchased fromSigma-Aldrich Chemical Co. (St. Louis, Mo.). All chemicals were usedwithout further purification. PBS buffer (1×, pH 7.4) was obtained fromInvitrogen (Carlsbad, Calif.). HPLC-grade ethyl acetate, hexanes,acetonitrile (CH₃CN), trifluoroacetic acid (TFA), and Millipore 18 mΩwater were used for purifications.

To a solution of 2-(tert-butoxycarbonylaminooxy)acetic acid (382 mg, 2mmol) in anhydrous dichloromethane (20 mL) was added sequentiallytriethylamine (307 μL, 2.2 mmol), N-(2-aminoethyl)maleimide-TFA salt(508 mg, 2 mmol), HOBT(306 mg, 2 mmol), and EDC (420 mg, 2.2 mmol).After being stirred for 24 hrs at room temperature, the reaction mixturewas diluted with ethyl acetate (50 mL) and washed with saturated sodiumbicarbonate solution (3×30 mL), water (30 mL), and brine (30 mL). Theorganic layer was dried over anhydrous magnesium sulfate and filtered.The filtrate was concentrated to a pale yellow solid, which was purifiedby column chromatography (70% ethyl acetate in hexanes) to give theproduct as a white powder (500 mg, 80% yield). ¹H-NMR (400 MHz, CDCl₃):δ 1.50 (s, 9H), 3.55 (tt, J1=6.0 Hz, J2=6.5 Hz, 2H), 3.77 (dd, J=7.6 Hz,2H), 4.30 (s, 2H), 6.3 (s, 2H).

A solution of 9.3 mg of Mal-AO-Boc in 1 mL of 3M HCl in methanol wasstirred at room temperature for 18 hours. Solvents were removed undervacuum to yield Mal-AO as a light yellow solid. (80% yield). ¹H-NMR (400MHz, DMSO-d₆): δ 3.27 CH₂ (t, J=4.0 Hz, 2H), 3.49 CH₂ (t, J=4.0 Hz, 2H),4.39 CH₂O (s, 2H), 7.00 CH═CH (s, 2H); m/z=214.07 for [M+H]⁺ (C₈H₁₂N₃O₄,Calculated MW=214.11))

The polypeptide was dissolved with freshly degassed PBS buffer (1×, pH7.4) at a concentration of approximately 1 mg/mL. The disulfide linkagein the polypeptide was reduced by the addition of dithiothreitol (DTT)solution in freshly degassed PBS buffer (1×, pH 7.4). The finalconcentration of DTT is 20 mM. The reaction mixture was vortexed for 2hours and eluted through a Zeba desalt spin column (Pierce Technologies)pre-equilibrated with degassed PBS buffer to remove excess of DTTreagent. The reduced polypeptide was collected, and the bifunctionalMal-AO compound was added (15 equivalents per equivalent of thepolypeptide) as a solution in DMSO. After being vortexed at roomtemperature overnight, the reaction mixture was purified with HighPerformance Liquid Chromatography (HPLC) (FIGS. 14A and 14B).

The HPLC purification was performed on a MiCHROM Magic C18AQ 5μ 200Acolumn (MiChrom Bioresources, Auburn, Calif.). Solvent A: H₂O (with 0.1%formic acid), Solvent B: CH₃CN (with 0.1% formic acid). Gradient: 5-100%B over 30 mins. The fractions containing desired product was combinedand neutralized with 100 mM ammonium bicarbonate solution, and thesolvents were removed by lyophilization to give the aminoxy-modifiedpolypeptide as a white solid.

ESI-TOF-MS analysis confirmed the presence of target product with theexpected molecular weights (calculated MW: 6964 Da, 8531 Da, and 7243Da, found: 6963 Da, 8532 Da, and 7244 Da for Z00477 (SEQ. ID No.4)-ONH₂, Z00342 (SEQ. ID No. 1)-ONH₂, and Z02891 (SEQ. ID No. 2)-ONH₂,respectively.

Methods: All reactions were performed either under a nitrogen atmosphereor in a crimp-top sealed vial purged with nitrogen prior to use.Kryptofix 222 (Aldrich) and K₂CO₃ (EMD Science) were purchased and usedas received. Optima™-grade acetonitrile was used as both HPLC andreaction solvents.

K¹⁸F (40 mCi·mL⁻¹ (1480 MBq·mL⁻¹) in purified water) was obtained fromIBA Molecular (Albany, N.Y.) and PETNET Solutions (Albany, N.Y.) andwere used as received. The [¹⁸F⁻] fluoride was first immobilized on aChromafix 30-PS-HCO3 anion exchange cartridge (ABX, Radeberg, Germany),then eluted into a drydown vessel with a 1 mL, 4:1 mixture ofacetonitrile:distilled, deionized H₂O (ddH₂O) containing Kryptofix K222(376 g·mol⁻¹, 8 mg, 2.13×10⁻⁵ mol) and potassium carbonate (138.2g·mol⁻¹, 2.1 mg, 1.52×10⁻⁵ mol). The solvent was removed under partialvacuum and a flow of nitrogen with gentle heating (˜45° C.) (˜15 min).The source vial and anion exchange cartridge were then washed with 0.5mL of acetonitrile containing K222 (8 mg) and the reaction mixture againbrought to dryness under partial vacuum and gentle heating (˜10 min).The reaction vessel was repressurized with nitrogen and the azeotropicdrydown repeated once with an additional 0.5 mL of acetonitrile.4-formyl-N,N,N-trimethylanilinium triflate (313.30 g·mol⁻¹, 3.1 mg,9.89×10⁻⁶ mol) was dissolved in 0.35 mL of anhydrous DMSO (Acros) andadded directly to the reaction vessel containing the K¹⁹F·K222, K₂CO₃.The reaction mixture was heated to 90° C. for 15 min and immediatelycooled and quenched with 3 mL of ddH₂O. This mixture was subsequentlypassed through a cation exchange cartridge (Waters SepPak Light AccellPlus CM), diluted to 10 mL with ddH₂O, and loaded onto a reverse phaseC18 SepPak (Waters SepPak Plus C18). The SepPak was flushed with 10 mLof ddH₂O then purged with 30 mL of air. [¹⁸F]4-fluorobenzaldehyde(¹⁸FBA), was eluted in 1.0 mL of methanol.

Separately, a high recovery vial (2 mL, National Scientific) was chargedwith either the Z00477-(SEQ. ID No. 3)-ONH₂ (0.35-0.5 mg), Z00342-(SEQ.ID No. 1)-ONH₂ (0.35-0.5 mg) or Z02891-(SEQ. ID No. 2)-ONH₂ (0.35-0.5mg). The solid was suspended in 25 μL of ddH₂O and 8 μL oftrifluoroacetic acid. 25 μL of ¹⁸FBA in methanol (see above) wastransferred to the reaction vial. The vessel was capped, crimped, placedin a heating block and maintained at 60° C. for 15 minutes; at whichpoint a small aliquot (<5 μL) was removed for analytical HPLC analysis.450 μL of ddH₂O with 0.1% TFA was used to dilute the solution to approx.500 μL in preparation for semi-preparative HPLC purification.¹⁸FB-Polypeptide was isolated and purified by semi-preparative HPLC. TheHPLC fraction containing the product (0.113 mCi/4.18 MBq) was diluted5:1 with ddH₂O and subsequently immobilized on a tC18 Plus Sep Pak(Waters). The SepPak was flushed first with 5 mL of ddH₂O then 30 mL ofair. ¹⁸FB-Polypeptide was isolated in a minimal amount of ethanol byfirst eluting the void volume (approx. 0.5 mL) followed by collecting250 to 300 μL of eluent in a separate flask. RP-HPLC analysis wasperformed on the isolated product in order to establish radiochemicaland chemical purity. Typically, 10 μL of a 0.1 μCi/μL solution wasinjected for post formulation analysis. Isolated radiochemical yieldsare indicated in Table 9 and are decay corrected from the addition ofpolypeptide to ¹⁸FBA and radiochemical purity of >99%. Alternatively,¹⁸F-labeled polypeptides were isolated by NAPS size exclusionchromatography by diluting the reaction mixture to approximately 0.5 mLwith 10 mM PBS and loading onto the gel. The ¹⁸F-labeled polypeptideswere isolated by eluting the column with 0.8 mL of 10 mM PBS and usedwithout further modification. These results are illustrated in Table 8,and FIG. 15.

TABLE 8 Yield isolated (decay HPLC Compound corrected)(%) RCP (%) Z00477(SEQ. ID No. 4) 0.6%/1.2%  95% Z00342 (SEQ. ID No. 1) 8.2% (10.7%) >99%Z02891 (SEQ. ID No. 2) 6.2% (7.6%) >99%

Analytical HPLC conditions used are as follows: Analysis performed on anHP Agilent 1100 with a G1311A QuatPump, G1313A autoinjector with 100 μLsyringe and 2.0 mL seat capillary, Phenomenex Gemini C18 column (4.6mm×150 mm), 5μ, 100 Å (S/N 420477-10), G1316A column heater, G1315A DADand Ramon Star—GABI gamma-detector. 95:5 ddH₂O:CH₃CN with 0.05% TFA,Solvent B: CH₃CN with 0.05% TFA. Gradient elution (1.0 mL·min⁻¹): 0 min.0% B, 1 min. 15% B, 21 min. 50% B, 22 min. 100% B, 26 min. 100% B, 27min. 0% B, 32 min. 0% B. or gradient elution (1.2 mL·min⁻¹): 0 min. 0%B, 1 min. 15% B, 10 min. 31% B, 10.5 min. 100% B, 13.5 min. 100% B, 14min. 0% B, 17 min. 0% B.

Semipreparative HPLC conditions used are as follows: Purification wasperformed on a Jasco LC with a DG-2080-54 4-line Degasser, an MX-2080-32Dynamic Mixer and two PU-2086 Plus Prep pumps, an AS-2055 PlusIntelligent autoinjector with large volume injection kit installed, aPhenomenex 5μ Luna C18(2) 100 Å, 250×10 mm, 5μ column with guard (S/N295860-1, P/N 00G-4252-NO), an MD-2055 PDA and a Carroll & RamseyAssociates Model 1055 Analogue Ratemeter attached to a solid-state SiPINphotodiode gamma detector. Gradient elution: 0 min. 5% B, 32 min. 20% B,43 min. 95% B, 46 min. 95% B, 49 min. 5% B, Solvent A: ddH₂O:CH₃CN with0.05% TFA, Solvent B: CH₃CN with 0.05% TFA.

In vivo studies were carried out with female CD-1 nude mice (CharlesRiver Labs, Hopkinton, Mass.) with an age range between 6 and 15 weeks.Mice were housed in a ventilated rack with food and water ad libitum anda standard 12 hour day-night lighting cycle. For xenografts, animalswere injected with 100 μl of cells in PBS. Cells were implantedsubcutaneously in the right hindquarter. Implantation was performedunder isoflurane anesthesia. For SKOV3, between 3×10⁶ to 4×10⁶ cellswere implanted in each mouse. Under these conditions, useable tumors(100 to 300 μg) were obtained in 3 to 4 weeks in greater than 80% ofanimals injected.

Mice were given tail-vein injections of ˜1 ug of ¹⁸F-labeled polypeptide(˜4 uCi/1 μg). Mice were placed in filter-paper lined cages untileuthanasia. Three mice were euthanized at each timepoint and tissues ofinterest dissected and counted on a Perkin Elmer Wallac Wizard 1480Gamma Counter. Data were collected for blood, kidney, liver, spleen,bone and injection site (tail). Urine from cages was pooled with thebladder and also counted. The remaining tissues were counted and the sumof all tissues plus urine for each animal was summed to provide thetotal injected dose. The percent injected dose for each organ wasdetermined based on this total, and organs were weighed fordetermination of the percent injected dose per gram, (% ID/g). Data isreported as mean value for all three mice in the timepoint with errorbars representing the standard deviation of the group.

The polypeptides underwent biodistribution studies in SKOV3 cellxenograft models. Four time points were taken over four hours (5, 30,120, and 240 minutes post-injection). Complete biodistribution data areincluded in table 12 (% ID/g Z02891 (SEQ. ID No. 2)-¹⁸F-fluorobenzyloxime in SKOV3 Tumor Bearing Mice) and table 13 (% ID/g Z00342 (SEQ. IDNo. 1) ¹⁸F-fluorobenzyl oxime in SKOV3 Tumor Bearing Mice). FIGS. 16, 17and 18 show the tumor, blood, tumor:blood, and clearance curves forthese tests.

The Z02891 (SEQ. ID No. 2) ¹⁸F-fluorobenzyl oxime polypeptide showsstrong tumor uptake in target-expressing SKOV3 tumors, with a value of17.47±2.89 (n=3) of the injected dose per gram of tissue at 240 minutespost-injection (PI). Tumor:blood ratios were approximately 3, 34, and128 at 30, 120, and 240 min post injection, respectively. The Z00342(SEQ. ID No. 1) ¹⁸F-fluorobenzyl oxime polypeptide shows strong tumoruptake in target-expressing SKOV3 tumors, with a value of 12.45±2.52(n=3) of the injected dose per gram of tissue at 240 minutes PI.Tumor:blood ratios were approximately 3, 32 and 53 at 30, 120 and 240min post injection, respectively.

The polypeptides exhibit a monoexponential clearance from the blood withhalf-lives of less than two minutes. This clearance of Z02891 (SEQ. IDNo. 2) is primarily mediated by the kidneys, with 0.95±0.07 (n=3)ID/organ at 240 min PI. Activity is secreted primarily in the urine.Polypeptide uptake in the spleen was minimal, and low uptake in theliver is observed, ca. 1.8% ID/organ (equivalent in value in mice to %ID/g) over the course of the study (four hours post injection).

TABLE 9 Z02891 (SEQ. ID No. 2) ¹⁸F-fluorobenzyl oxime uptake (% ID/g) inSKOV-3 tumor bearing mice 5 Minutes 30 Minutes 120 Minutes 240 MinutesBlood 9.23 ± 0.68 2.91 ± 0.23 (n = 3) 0.40 ± 0.07 0.14 ± 0.02 (n = 3) (n= 3) (n = 3) Tumor 2.39 ± 1.13 8.91 ± 2.09(n = 3) 13.47 ± 3.61 17.47 ±2.89 (n = 3) (n = 3) (n = 3) Liver 4.68 ± 0.45 3.85 ± 0.95 (n = 3) 1.57± 0.42 1.59 ± 0.83 (n = 3) (n = 3) (n = 3) Kidney 72.42 ± 15.61 35.02 ±5.76(n = 3) 5.22 ± 0.65 2.49 ± 0.17 (n = 3) (n = 3) (n = 3) Spleen 3.04± 1.15 1.46 ± 0.05 (n = 3) 0.37 ± 0.01 0.26 ± 0.04 (n = 3) (n = 3) (n =3)

TABLE 10 Z00342 (SEQ. ID No. 1) ¹⁸F-fluorobenzyl oxime uptake (% ID/g)in SKOV-3 tumor bearing mice 5 Minutes 30 Minutes 120 Minutes 240Minutes Blood 7.38 ± 0.72 1.76 ± 0.09 (n = 3) 0.33 ± 0.08 0.87 ± 0.98 (n= 3) (n = 3) (n = 3) Tumor 2.54 ± 0.00 4.97 ± 3.14 (n = 3) 10.30 ± 1.0812.45 ± 2.52 (n = 2) (n = 3) (n = 3) Liver 8.29 ± 0.41 6.94 ± 0.92 (n =3) 2.54 ± 1.44 1.41 ± 0.35 (n = 3) (n = 3) (n = 3) Kidney 78.93 ± 2.9330.94 ± 4.93 (n = 3) 10.75 ± 2.17 4.91 ± 0.63 (n = 3) (n = 3) (n = 3)Spleen 3.85 ± 0.51 1.77 ± 0.34 (n = 3) 0.47 ± 0.08 0.23 ± 0.05 (n = 3)(n = 3) (n = 3)

All reactions are performed either under a nitrogen atmosphere or in acrimp-top sealed vial purged with nitrogen. Optima™-grade acetonitrileis used as both HPLC and reaction solvents.

[¹²³I]4-iodobenzaldehyde (¹²³I BA) is added to a high recovery vial (2mL, National Scientific) containing the polypeptide-ONH₂ (Z02891, SEQ.ID No. 2), 0.35-0.5 mg). The reaction commences by dissolving thepolypeptide in 25 μL of ddH₂O and adding 8 μL of trifluoroacetic acidfollowed by the addition of ¹²³IIBA in methanol. The vessel is capped,crimped, placed in a heating block and maintained at 60° C. for 15minutes; removing a small aliquot (<5 μL) for analytical HPLC analysisis done to assess the status of the reaction. The reaction mixture isdiluted to a minimum 1:1 mixture of ddH₂O:Acetonitrile mixturecontaining 0.1% TFA in preparation for semi-preparative HPLCpurification. ¹²³IB-Polypeptide is isolated and purified bysemi-preparative HPLC or NAPS size exclusion chromatography. The HPLCfraction containing the product is further diluted (5:1) with ddH₂O andsubsequently immobilized on a tC18 Plus Sep Pak (Waters). Flushing theSepPak first with 5 mL of ddH₂O then 30 mL of air gives the¹²³IB-Polypeptide in a minimal amount of ethanol by first eluting thevoid volume (approx. 0.5 mL) followed by collecting 250 to 300 μL ofeluent in a separate flask. RP-HPLC analysis is performed on theisolated product to establish radiochemical and chemical purity.

Polypeptide Z00477 (SEQ. ID 3) was labeled with Ga, specifically ⁶⁷Ga,after a NOTA (1,4,7-triazacyclononane-N,N′,N″-triacetic acid) chelatorwas conjugated to the polypeptide. (FIG. 19)

Bioconjugation of Mal-NOTA to polypeptide molecules was accomplished asfollows. The polypeptide was dissolved with freshly degassed PBS buffer(1×, pH 7.4) at a concentration of approximately 1 mg/mL. The disulfidelinkage in the polypeptide was reduced by the addition of DTT solutionin freshly degassed PBS buffer (1×, pH 7.4). The final concentration ofDTT was 20 mM. The reaction mixture was vortexed for 2 hours and passedthrough a Zeba desalt spin column (Pierce Technologies) pre-equilibratedwith degassed PBS buffer (1×, pH 7.4) to remove excess of DTT reagent.The eluted reduced polypeptide molecule was collected, and thebifunctional compound mal-NOTA was added (15 equivalents per equivalentof the polypeptide) as a solution in DMSO, and the mixture was vortexedat room temperature. The reaction was allowed to proceed overnight toensure the complete conversion of the polypeptide molecules.

The HPLC purification was performed on a MiCHROM Magic C18AQ 200A column(MiChrom Bioresources, Auburn, Calif.). Solvent A: H₂O (with 0.1% formicacid), Solvent B: CH₃CN (with 0.1% formic acid). Gradient: 5-100% B over30 mins. (FIG. 20A)

The fractions containing desired product were combined and neutralizedwith 100 mM ammonium bicarbonate solution, and the solvents were removedby lyophilization to give the conjugated polypeptide as a white solid.

LC-MS analysis of the purified product confirmed the presence of thedesired product, and the MW suggested that only one NOTA chelator wasadded to the polypeptide construct (calculated MW: 7504 Da, found: 7506Da for Z00477 (SEQ. ID No. 3)-NOTA). (FIG. 20B)

Radiolabeling was subsequently accomplished as follows: 25 μl HEPESsolution (63 mM) was initially added to a screw top vial followed by 10μl ⁶⁷GaCl₃ (GE Healthcare) in 40.5 MBq of 0.04M HCl. 30 μg (MW=7506,4.0×10⁻⁹ mol) of the NOTA Z00477 (SEQ. ID No. 3) in 30 μl H₂O was thenadded to the reaction mixture to give a final NOTA Z00477 (SEQ. ID No.3) concentration of 61 μM with a pH of 3.5-4.0. The reaction vial wassealed and the reaction maintained at ambient temperature. Reverse phaseHPLC analysis of the crude reaction mixture determined the radiochemicalpurity of the ⁶⁷Ga-NOTA Z00477 (SEQ. ID No. 3) was determined to be 95%by HPLC after 2 hours at room temperature. (FIG. 21) The ⁶⁷Ga-NOTAZ00477 (SEQ. ID No. 3) was purified by HPLC after a reaction time of 1day. 22 MBq of ⁶⁷Ga-NOTA Z00477 (SEQ. ID No. 3) was injected onto theHPLC for the purification. 15 MBq of the ⁶⁷Ga labeled product wasobtained from the purification (radiochemical yield=68%). HPLC solventswere removed under vacuum to give a solution with an approximate volumeof 0.5 mL. Approximately 1.45 mL of Dulbecco's phosphate buffered salinewas then added to give a final solution at pH 6-6.5 with a radioactivityconcentration of 7.7 MBq/mL. Purified, formulated ⁶⁷Ga-NOTA Z00477 (SEQ.ID No. 3) was found to be stable for at least 2 hr at room temperature.(RCP=96% by HPLC) (FIG. 22).

Analytical HPLC conditions used are as follows: A Grace Vydac C₄ protein5 micron, 300 Å, 4.6×250 mm HPLC column. Solvent A=95/5 H₂O/MeCN in0.05% trifluoroacetic acid (TFA) Solvent B=95/5 CH₃CN/H₂O in 0.05% TFA.HPLC gradient (Min/% B): 0/0, 4/20, 16/60, 20/100, 25/100, 26/0.

Semi-preparative HPLC conditions used are as follows: Column: GraceVydac C4 protein 5 micron, 300 Å, 4.6×250 mm. Solvent A=95/5 H₂O/MeCN in0.05% trifluoroacetic acid (TFA) Solvent B=95/5 CH₃CN/H₂O in 0.05% TFA.HPLC gradient (Min/% B): 0/0, 4/20, 16/60, 20/100, 25/100, 26/0.

While only certain features of the invention have been illustrated anddescribed herein, many modifications and changes will occur to thoseskilled in the art. It is, therefore, to be understood that the appendedclaims are intended to cover all such modifications and changes as fallwithin the true spirit of the invention.

1.-10. (canceled)
 11. An imaging compound comprising: an isolatedpolypeptide and a radioisotope coupled to the peptide through abifunctional linker molecule, wherein the radioisotope is coupled to thebifunctional linker via an aminoxy group, wherein the isolatedpolypeptide comprises SEQ. ID No. 1 or SEQ. ID No. 2, and wherein theisolated polypeptide binds specifically to HER2.
 12. The compound ofclaim 11, wherein the radioisotope is ¹⁸F.
 13. The compound of claim 12,wherein the radioisotope is ^(99m)Tc.
 14. The compound of claim 11,wherein the bifunctional aminoxy linker is a tert-butyl2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylamino)-2-oxoethoxycarbamatelinker.
 15. The compound of claim 11, wherein the radioisotope isconjugated to the isolated polypeptide via the bifunctional aminoxylinker at the N-terminus of the isolated polypeptide.
 16. The compoundof claim 15, wherein the radioisotope is ¹⁸F.
 17. An imaging compoundcomprising: an isolated polypeptide comprising SEQ. ID No. 2 conjugatedwith a radioisotope via a tert-butyl2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylamino)-2-oxoethoxycarbamatelinker, wherein the isolated polypeptide binds specifically to HER2. 18.The method of claim 17, wherein the radioisotope is ¹⁸F.
 19. The methodof claim 18, wherein the radioisotope is ^(99m)Tc.